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- EMDB-25678: Structure of dimeric phosphorylated Pediculus humanus (Ph) PINK1 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-25678
TitleStructure of dimeric phosphorylated Pediculus humanus (Ph) PINK1 with kinked alpha-C helix in chain B
Map dataDimer map (locally filtered). Generated from 3D variability analysis and local refinement of the dimer. Initial dimer was generated from local refinement of a dimer from the dodecamer.
Sample
  • Complex: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1
    • Protein or peptide: Serine/threonine-protein kinase PINK1, putative
Function / homology
Function and homology information


positive regulation of mitochondrial fission / autophagy / regulation of apoptotic process / mitochondrial outer membrane / mitochondrial inner membrane / non-specific serine/threonine protein kinase / protein kinase activity / phosphorylation / protein serine/threonine kinase activity / ATP binding ...positive regulation of mitochondrial fission / autophagy / regulation of apoptotic process / mitochondrial outer membrane / mitochondrial inner membrane / non-specific serine/threonine protein kinase / protein kinase activity / phosphorylation / protein serine/threonine kinase activity / ATP binding / metal ion binding / cytosol
Similarity search - Function
Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase Pink1, mitochondrial
Similarity search - Component
Biological speciesPediculus humanus corporis (human body louse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsGan ZY / Leis A / Dewson G / Glukhova A / Komander D
Funding support United States, 2 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia) United States
Michael J. Fox Foundation United States
CitationJournal: Nature / Year: 2022
Title: Activation mechanism of PINK1.
Authors: Zhong Yan Gan / Sylvie Callegari / Simon A Cobbold / Thomas R Cotton / Michael J Mlodzianoski / Alexander F Schubert / Niall D Geoghegan / Kelly L Rogers / Andrew Leis / Grant Dewson / Alisa ...Authors: Zhong Yan Gan / Sylvie Callegari / Simon A Cobbold / Thomas R Cotton / Michael J Mlodzianoski / Alexander F Schubert / Niall D Geoghegan / Kelly L Rogers / Andrew Leis / Grant Dewson / Alisa Glukhova / David Komander /
Abstract: Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's disease. PINK1 has many unique features that enable it to phosphorylate ...Mutations in the protein kinase PINK1 lead to defects in mitophagy and cause autosomal recessive early onset Parkinson's disease. PINK1 has many unique features that enable it to phosphorylate ubiquitin and the ubiquitin-like domain of Parkin. Structural analysis of PINK1 from diverse insect species with and without ubiquitin provided snapshots of distinct structural states yet did not explain how PINK1 is activated. Here we elucidate the activation mechanism of PINK1 using crystallography and cryo-electron microscopy (cryo-EM). A crystal structure of unphosphorylated Pediculus humanus corporis (Ph; human body louse) PINK1 resolves an N-terminal helix, revealing the orientation of unphosphorylated yet active PINK1 on the mitochondria. We further provide a cryo-EM structure of a symmetric PhPINK1 dimer trapped during the process of trans-autophosphorylation, as well as a cryo-EM structure of phosphorylated PhPINK1 undergoing a conformational change to an active ubiquitin kinase state. Structures and phosphorylation studies further identify a role for regulatory PINK1 oxidation. Together, our research delineates the complete activation mechanism of PINK1, illuminates how PINK1 interacts with the mitochondrial outer membrane and reveals how PINK1 activity may be modulated by mitochondrial reactive oxygen species.
History
DepositionDec 10, 2021-
Header (metadata) releaseJan 12, 2022-
Map releaseJan 12, 2022-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7t4k
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25678.png

Downloads & links

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Map

FileDownload / File: emd_25678.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDimer map (locally filtered). Generated from 3D variability analysis and local refinement of the dimer. Initial dimer was generated from local refinement of a dimer from the dodecamer.
Voxel sizeX=Y=Z: 0.78 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.41887543 - 0.75837994
Average (Standard dev.)0.00030716392 (±0.008603486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 343.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.780.780.78
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z343.200343.200343.200
α/β/γ90.00090.00090.000
start NX/NY/NZ253841
NX/NY/NZ999350
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.4190.7580.000

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Supplemental data

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Mask #1

Fileemd_25678_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Dimer map (unsharpened).

Fileemd_25678_additional_1.map
AnnotationDimer map (unsharpened).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer half map.

Fileemd_25678_half_map_1.map
AnnotationDimer half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Dimer half map.

Fileemd_25678_half_map_2.map
AnnotationDimer half map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dodecameric phosphorylated Pediculus humanus (Ph) PINK1

EntireName: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1
Components
  • Complex: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1
    • Protein or peptide: Serine/threonine-protein kinase PINK1, putative

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Supramolecule #1: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1

SupramoleculeName: Dodecameric phosphorylated Pediculus humanus (Ph) PINK1
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Dodecamer was stabilised by hydrogen peroxide treatment prior to phosphorylation and purification by SEC
Source (natural)Organism: Pediculus humanus corporis (human body louse)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pOPINK

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Macromolecule #1: Serine/threonine-protein kinase PINK1, putative

MacromoleculeName: Serine/threonine-protein kinase PINK1, putative / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Pediculus humanus corporis (human body louse)
Molecular weightTheoretical: 53.053602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GPSGLLTKDD ELEGICWEIR EAVSKGKWND SESENVEQLQ AANLDELDLG EPIAKGCNAV VYSAKLKNVQ SNKLAHQLAV KMMFNYDVE (SEP)NSTAILKAM YRETVPAMSY FFNQNLFNIE NISDFKIRLP PHPNIVRMYS VFADRIPDLQ CNKQLYP EA LPPRINPEGS ...String:
GPSGLLTKDD ELEGICWEIR EAVSKGKWND SESENVEQLQ AANLDELDLG EPIAKGCNAV VYSAKLKNVQ SNKLAHQLAV KMMFNYDVE (SEP)NSTAILKAM YRETVPAMSY FFNQNLFNIE NISDFKIRLP PHPNIVRMYS VFADRIPDLQ CNKQLYP EA LPPRINPEGS GRNMSLFLVM KRYDCTLKEY LRDKTPNMRS SILLLSQLLE AVAHMNIHNI SHRDLKSDNI LVDLSEGD A YPTIVITDFG CCLCDKQNGL VIPYRSEDQD KGGNRALMAP EIANAKPGTF SWLNYKKSDL WAVGAIAYEI FNIDNPFYD KTMKLLSKSY KEEDLPELPD TIPFIIRNLV SNMLSRSTNK RLDCDVAATV AQLYLWAPSS WLKENYTLPN SNEIIQWLLC LSSKVLCER DITARNKTNT MSESVSKAQY KGRRSLPEYE LIASFLRRVR LHLVRKGLKW IQELHIYN

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
25.0 mMTristrisaminomethane
150.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 1.25 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 212979
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6eqi


Chain - Chain ID: C
RefinementSpace: REAL
Output model

PDB-7t4k:
Structure of dimeric phosphorylated Pediculus humanus (Ph) PINK1 with kinked alpha-C helix in chain B

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